小鼠假体周围关节 白色念珠菌 感染模型
Summary
由危险病原体引起的假体周围关节感染 （PJI） 在临床骨科中很常见。现有的动物模型无法准确模拟PJI的实际情况。在这里，我们建立了一个 白色念珠菌 生物膜相关的PJI小鼠模型，以研究和开发PJI的新疗法。
Abstract
假体周围关节感染 （PJI） 是由 白色念珠菌 （C. albicans） 引起的常见感染之一， 越来越受到外科医生和科学家的关注。通常，可以在感染部位形成可以保护 白色念珠菌 免受抗生素和免疫清除的生物膜。手术包括切除受感染的植入物、清创术、抗菌治疗和再植入术是治疗 PJI 的金标准。因此，建立动物PJI模型对于PJI新药或新疗法的研发具有重要意义。在这项研究中，将一根光滑的镍钛合金丝（骨科诊所广泛使用的植入物）插入 C57BL/6 小鼠的股骨关节，然后沿线将 白色念珠菌 接种到关节腔中。14天后，在扫描电子显微镜（SEM）下在植入物表面观察到成熟和厚厚的生物膜。在感染关节标本的H&E染色中发现骨小梁明显减少。综上所述，建立了具有操作简便、成功率高、重复性高、临床相关性高等优点的小鼠PJI模型。这有望成为白色 念珠菌 生物膜相关PJI预防临床研究的重要模型。
Introduction
白色念珠菌（白色念珠菌）共存于人体的许多部位1，这也是最常见的机会性病原体，可引起危及生命的侵袭性真菌感染，尤其是在免疫功能低下的患者中2,3。白色念珠菌可以作为多态性真菌在酵母和菌丝体状态之间转化。菌丝体状态表现出更高的毒力、更强的粘附性以及对细胞和组织的侵袭 4,5。此外，白色念珠菌可以在假牙、导管和支架等生物医学材料的表面形成生物膜1,6,7。生物膜致密的三维结构限制了抗真菌药物的浸润，表达耐药基因，下调真菌细胞的代谢以抵抗免疫系统清除6,7。因此，与生物膜相关的感染在临床中非常具有挑战性8.
金黄色葡萄球菌、凝固酶阴性葡萄球菌和肠杆菌是引起PJI9的主要病原体。虽然真菌PJI的发生率相对较低（约1%）10，但真菌PJI的治疗成本较高11，治疗周期较长11，治疗成功率低于细菌PJI10。近年来，真菌PJI的发病率逐年增加10。念珠菌PJI占真菌PJI 10,12的77%-84%，白色念珠菌是念珠菌中最常见（54%）。因此，需要研究真菌PJI。
目前，PJI 通过翻修手术进行治疗，包括 （1） 移除受感染的植入物、（2） 清创术、（3） 抗菌治疗和 （4） 再植入。彻底清创后，放置含有骨水泥的抗生素，并对患者进行全身抗生素治疗 6 周以上，以有效控制感染，然后再放置新的植入物13。然而，这种方法不能完全消除组织中的病原体，并且用长期抗菌治疗治疗的复发性感染极有可能在耐药菌株中发展14,15,16。
建立PJI的动物模型对于PJI新药或疗法的研发具有重要意义。在PJI的发展中，假体周围形成大的死腔，导致血肿的形成，血肿进一步阻塞周围组织的血液供应并损害抗生素的作用11,15。由于难以模拟假体的周围环境，传统的动物模型无法准确模拟PJI17,18的实际情况。
在本文中，通过使用临床上广泛使用的钛镍丝来模拟关节植入物，在小鼠中构建了白色念珠菌生物膜相关的PJI模型19,20。该PJI模型具有操作简单、成功率高、重复性高、临床相关性高等优点。有望成为研究白色念珠菌生物膜相关PJI防治的重要模型。
Protocol
这些动物是从习交通大学购买的。所有动物实验程序均经习交通大学动物伦理学委员会批准（批准号：SCXK [陕西] 2021-103）。将小鼠饲养一周，每个笼子饲养5只小鼠。他们被允许免费获得食物和水。在进行研究之前，将动物保持在室温（RT;24°C±1°C）和光/暗循环（12小时/12小时）下。 1.缓冲液和设备准备 白色念珠菌 细胞培养将酵母提取物蛋白?…
Representative Results
将样品转移到平板培养基上并在孵育过夜后计数菌落通常用于评估病灶附近的局部病原体载量22,23。在我们的研究中，肝脏、肾脏和脾脏样本的微生物培养呈阴性，表明本研究中的模型仅导致小鼠的局部感染而不是全身感染23。 植入物的SEM图像如 图2所示。在空白植入物组中，没有白色 念<…
Discussion
手术过程中手术器械或手术环境的污染引起的感染是大多数植入物感染的主要原因24,25,26,27。因此，本研究构建了小鼠白色念珠菌生物膜相关PJI模型。与传统的PJI模型相比，以悬浮在盐水中的无菌不锈钢颗粒为植入物，本研究使用常用的植入物材料镍钛合金丝来模拟白色念珠菌、植入物?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
感谢陕西省自然科学基金（批准号：2021SF-118）和国家自然科学基金（批准号：81973409,82204631）的财政支持。
Materials
| 0.5 Mactutrius turbidibris | Shanghai Lujing Technology Co., Ltd | 5106063 | |
| 4 °C refrigerator | Electrolux (China) Electric Co., Ltd | ESE6539TA | |
| Agar | Beijing Aoboxing Bio-tech Co., Ltd | 01-023 | |
| Analytical balances | Shimadzu | ATX124 | |
| Autoclaves Sterilizer | SANYO | MLS-3750 | |
| Carbenicillin | Amresco | C0885 | |
| Eclipse Ci Nikon upright optical microscope | Nikon | Eclipse Ts2-FL | |
| Glucose | Macklin | D823520 | |
| Inoculation ring | Thermo Scientific | 251586 | |
| Isoflurane | RWD | 20210103 | |
| NaCl | Xi'an Jingxi Shuanghe Pharmaceutical Co., Ltd | 20180108 | |
| Paraformaldehyde | Beyotime Biotechnology | P0099 | |
| Peptone | Beijing Aoboxing Bio-tech Co., Ltd | 01-001 | |
| RWD R550 multi-channel small animal anesthesia machine | RWD | R550 | |
| SEM | Hitachi | TM-1000 | |
| Temperature incubator | Shanghai Zhichu Instrument Co., Ltd | ZQTY-50N | |
| Ultrapure water water generator | Heal Force | NW20VF | |
| Ultrasound machine | Do-Chrom | DS10260D | |
| Yeast extract | Thermo Scientific Oxoid | LP0021B |
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Cite This Article
Yang, C., Zhang, J., Mo, F., Zhang, P., Li, Q., Zhang, J. A Periprosthetic Joint Candida albicans Infection Model in Mouse. J. Vis. Exp. (204), e65263, doi:10.3791/65263 (2024).